We seek to understand the molecular mechanisms of visual excitation and light adaptation in vertebrate photoreceptors. Development of a molecular understanding of the photophysiology of retinal photoreceptors will provide crucial baseline data that should prove useful for the understanding of abnormalities in retinal degenerations due to disease or aging. Numerous reactions occur at the cytoplasmic surface of the rod outer segment (ROS) disk membrane in response to light excitation. We will study the light controlled binding reactions on the membrane surface that change the membrane surface charge using an electrophoretic laser light scattering technique. Protein binding constants, interactions between proteins, and the ion and nucleotide modulation of these surface binding reactions will be studied. A parallel line of investigation will focus on the coupling of light dependent reactions with the small "signal" molecules: calcium, protons, nucleotides and inositol phosphates. Initial emphasis will be placed on investigating the regulation of cell calcium by inositol tris-phosphate (IP3) which will be studied in permeabilized ROS and ROS with the inner segments attached (ROS-RIS). Effects of light on phosphatidyl inositol (PI) metabolism in ROS-RIS and whole retina will be investigated as well as the phosphorylation of proteins controlled by the diglyceride metabolite of PI and calcium. The roles of calcium, magnesium and protons in light dependent reactions will be studied using ion selective electrodes. Light scattering and other direct monitors will be used to follow GTP dependent protein binding, GTPase activity, ATP dependent disk membrane ion gradients, and ATP dependent protein binding to the membrane simultaneously with ion selective electrode measurements. Nucleotide levels and the rates of nucleotide turnover reactions will be measured nondestructively in whole living retinas by phosphorus NMR. Proton NMR will be adapted to study the levels and turnover reactions of the major protonated metabolites in the retina.